Network pharmacology combined with experimental verification to explore the potential mechanism of naringenin in the treatment of cervical cancer.

Cervical cancer is the second leading cause of morbidity and mortality in women worldwide. Traditional treatment methods have become limited. Naringenin, a flavonoid abundant in various fruits and herbal medicines, has demonstrated anti-tumor properties among other effects. This research undertook to elucidate the mechanism of naringenin in the context of cervical cancer treatment by leveraging network pharmacology and performing experimental validation. Initial steps involved predicting potential naringenin targets and subsequently screening for overlaps between these targets and those related to cervical cancer, followed by analysis of their interrelationships. Molecular docking was subsequently utilized to verify the binding effect of the central target. Within the framework of network pharmacology, it was discovered that naringenin might possess anti-cancer properties specific to cervical cancer. Following this, the anti-tumor effects of naringenin on Hela cell viability, migration, and invasion were assessed employing CCK-8, transwell, wound healing assays, and western blotting. Experimental data indicated that naringenin attenuates the migration and invasion of Hela cells via downregulation EGFR/PI3K/AKT signaling pathway. Thus, our findings suggest that naringenin has therapeutic impacts on cervical cancer via multiple mechanisms, primarily by inhibiting the migration and invasion through the EGFR/PI3K/AKT/mTOR pathway. This study offers fresh insights for future clinical studies.

The ferroptosis signature predicts the prognosis and immune microenvironment of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a cancer with a high metastatic rate and poor prognosis. Growing studies suggest that ferroptosis take part in the development of tumours. At the same time, the connection between ferroptosis-related genes (FRGs) and the prognosis of NPC remains unclear. In this study, we explored the dysregulated FRGs between normal control and tumour samples of NPC. Firstly, 14 of 36 differentially expressed FRGs were identified in NPC tissues compared to normal tissues, among which ABCC1, GLS2, CS and HMGCR were associated with poor prognosis for patients. The four ferroptosis genes were used for consensus cluster analysis and two risk-related FRGs (ABCC1 and GLS2) were used in a risk model. The ROC curve revealed the good predictive performance of this risk signature. Multivariate analysis revealed that risk score and intratumoral TILs were independent risk factors linked to prognosis. Additionally, our results suggested that the risk signature was attached to the immune microenvironment. Moreover, the NPC patients with high risk were sensitive to chemotherapeutic drugs including axitinib, docetaxel, embelin, epothilone.B, parthenolide, thapsigargin, tipifarnib, vinorelbine. Finally, the expression of ABCC1 and GLS2 was validated in NPC tissues using immunohistochemistry. Together, these results revealed ferroptosis may be a potential biomarker in NPC and representing a promising future direction in prognosis and therapeutic strategy for the treatment of NPC.

Phosphorylation of nucleolin is indispensable to its involvement in the proliferation and migration of non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is one of the mostly deadly malignancies in the world. Nucleolin is a multifunctional protein that mainly regulates ribosome biogenesis but also has other functions including modulating the transcription of mRNAs and repressing RNA polymerase II. Nucleolin is overexpressed in various cancer cells, including NSCLC cells. It can confer resistance to apoptosis and promote cell migration and blood vessel formation by directly taking part in various tumor signal transduction pathways. The activities of nucleolin are regulated mainly by intracellular localization and post-translational modifications, including phosphorylation, glycosylation, methylation, and ADP-ribosylation. Phosphorylation of nucleolin (P-nucleolin) in NSCLC cells is still not well characterized. In the present study, the levels of nucleolin and P-nucleolin were examined in lung tissue and cells and it was demonstrated that levels of the two forms of nucleolin were significantly increased in NSCLC compared with non-cancerous tissues and cells. In addition, it was demonstrated that high expression levels of nucleolin and P-nucleolin were significantly associated with poor overall survival of NSCLC patients. Doxorubicin (DOX) is a type of anthracycline that has been used in the treatment of various types of cancer, including NSCLC. Upregulation of nucleolin through exogenous expression of nucleolin promoted A549 cell proliferation and migration, while downregulation of nucleolin through expression of small interfering RNA-nucleolin attenuated A549 cell proliferation and migration. Following stimulation with DOX, A549 cell proliferation and migration decreased and the expression of P-nucleolin also decreased. In order to investigate whether P-nucleolin is indispensable to the proliferation and migration of NSCLC cells, a plasmid encoding mutant nucleolin, in which the phosphorylation site at threonine-76 was mutated to alanine, was constructed. Compared with the A549 cells transfected with wild-type nucleolin, P-nucleolin expression and cell proliferation and migration were significantly decreased in A549 cells transfected with mutant nucleolin. These results indicate that targeting P-nucleolin may be a promising strategy for treating NSCLC patients.

Pull-down combined with proteomic strategy reveals functional diversity of synaptotagmin I.

Synaptotagmin I (Syt I) is most abundant in the brain and is involved in multiple cellular processes. Its two C2 domains, C2A and C2B, are the main functional regions. Our present study employed a pull-down combined with proteomic strategy to identify the C2 domain-interacting proteins to comprehensively understand the biological roles of the C2 domains and thus the functional diversity of Syt I. A total of 135 non-redundant proteins interacting with the C2 domains of Syt I were identified. Out of them, 32 and 64 proteins only bound to C2A or C2B domains, respectively, and 39 proteins bound to both of them. Compared with C2A, C2B could bind to many more proteins particularly those involved in synaptic transmission and metabolic regulation. Functional analysis indicated that Syt I may exert impacts by interacting with other proteins on multiple cellular processes, including vesicular membrane trafficking, synaptic transmission, metabolic regulation, catalysis, transmembrane transport and structure formation, etc. These results demonstrate that the functional diversity of Syt I is higher than previously expected, that its two domains may mediate the same and different cellular processes cooperatively or independently, and that C2B domain may play even more important roles than C2A in the functioning of Syt I. This work not only further deepened our understanding of the functional diversity of Syt I and the functional differences between its two C2 domains, but also provided important clues for the further related researches.

Nitroproteins in Human Astrocytomas Discovered by Gel Electrophoresis and Tandem Mass Spectrometry.

Protein tyrosine nitration is involved in the pathogenesis of highly fatal astrocytomas, a type of brain cancer. To understand the molecular mechanisms of astrocytomas and to discover new biomarkers/therapeutic targets, we sought to identify nitroproteins in human astrocytoma tissue. Anti-nitrotyrosine immunoreaction-positive proteins from a high-grade astrocytoma tissue were detected with two-dimensional gel electrophoresis (2DGE)-based nitrotyrosine immunoblots, and identified with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Fifty-seven nitrotyrosine immunopositive protein spots were detected. A total of 870 proteins (nitrated and non-nitrated) in nitrotyrosine-immunopositive 2D gel spots were identified, and 18 nitroproteins and their 20 nitrotyrosine sites were identified with MS/MS analysis. These nitroproteins participate in multiple processes, including drug-resistance, signal transduction, cytoskeleton, transcription and translation, cell proliferation and apoptosis, immune response, phenotypic dedifferentiation, cell migration, and metastasis. Among those nitroproteins that might play a role in astrocytomas was nitro-sorcin, which is involved in drug resistance and metastasis and might play a role in the spread and treatment of an astrocytoma. Semiquantitative immune-based measurements of different sorcin expressions were found among different grades of astrocytomas relative to controls, and a semiquantitative increased nitration level in high-grade astrocytoma relative to control. Nitro-ß-tubulin functions in cytoskeleton and cell migration. Semiquantitative immunoreactivity of ß-tubulin showed increased expression among different grades of astrocytomas relative to controls and semiquantitatively increased nitration level in high-grade astrocytoma relative to control. Each nitroprotein was rationalized and related to the corresponding functional system to provide new insights into tyrosine nitration and its potential role in the pathogenesis of astrocytoma formation. Graphical Abstract ᅟ.

Identification of Glioblastoma Phosphotyrosine-Containing Proteins with Two-Dimensional Western Blotting and Tandem Mass Spectrometry.

To investigate the presence of, and the potential biological roles of, protein tyrosine phosphorylation in the glioblastoma pathogenesis, two-dimensional gel electrophoresis- (2DGE-) based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. MS/MS and Mascot analyses were used to determine the phosphotyrosine sites of each phosphopeptide. Protein domain and motif analysis and systems pathway analysis were used to determine the protein domains/motifs that contained phosphotyrosine residue and signal pathway networks to clarify the potential biological functions of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress, stress response, and cell migration. Those data show 2DGE-based Western blotting, MS/MS, and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma.

Proteomic and functional profiles of a follicle-stimulating hormone positive human nonfunctional pituitary adenoma.

Nonfunctional pituitary adenoma (NFPA) is highly heterogeneous with different hormone-expressed subtypes in NFPA tissues including follicle-stimulating hormone (FSH) positive, luteinizing hormone-positive, FSH/luteinizing hormone-positive, and negative types. To analyze in-depth the variations in the proteomes among different NFPA subtypes for our long-term goal to clarify molecular mechanisms of NFPA and to detect tumor biomarker for personalized medicine practice, a reference map of proteome of a human FSH-expressed NFPA tissue was described here. 2DE and PDQuest image analysis were used to array each protein. MALDI-TOF PMF and human Swiss-Prot databases with MASCOT search were used to identify each protein. A good 2DE pattern with high level of between-gel reproducibility was attained with an average positional deviation 1.98 ± 0.75 mm in the IEF direction and 1.62 ± 0.68 mm in the SDS-PAGE direction. Approximately 1200 protein spots were 2DE-detected and 192 redundant proteins that were contained in 141 protein spots were PMF-identified, representing 107 nonredundant proteins. Those proteins were located in cytoplasm, nucleus, plasma membrane, extracellular space, and so on, and those functioned in transmembrane receptor, ion channel, transcription/translation regulator, transporter, enzyme, phosphatase, kinase, and so on. Several important pathway networks were characterized from those identified proteins with DAVID and Ingenuity Pathway Analysis systems, including gluconeogenesis and glycolysis, mitochondrial dysfunction, oxidative stress, cell-cycle alteration, MAPKsignaling system, immune response, TP53-signaling, VEGF-signaling, and inflammation signaling pathways. Those resulting data contribute to a functional profile of the proteome of a human FSH-positive NFPA tissue, and will serve as a reference for the heterogeneity analysis of NFPA proteomes.

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity.

BACKGROUND: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity. RESULTS: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+ and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed. CONCLUSIONS: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

Identification of the proteomic variations of invasive relative to non-invasive non-functional pituitary adenomas.

The incomplete surgery section of invasive non-functional pituitary adenomas (NFPAs) carries the increased risks of complications and requires adjuvant radiotherapy and medications. It is necessary to clarify the molecular mechanisms and markers of invasiveness to guide the management of NFPA patients. The study aimed to proteomic variations of invasive and non-invasive NFPAs and sought the protein markers for invasive NFPAs. Invasive (n = 4) and non-invasive (n = 4) NFPA tissues were analyzed (n = 3-5/each tissue) with 2DE and PDQuest software. Twenty-four high-resolution 2DE gels were quantitatively compared to determine differentially expressed proteins (DEPs) between invasive and non-invasive NFPAs. Approximately 1200 protein spots were detected in each 2DE map, and 103 differential spots (64 upregulated and 39 downregulated) were identified. Among those 103 differential spots, 57 DEPs (30 upregulated and 27 downregulated) were characterized with peptide mass fingerprint and MS/MS. Gene-ontology (GO) and ingenuity pathway analyses of those DEPs revealed pathway networks including mitochondrial dysfunction, oxidative stress, mitogen-activated protein kinase signaling abnormality, TR/RXR activation, proteolysis abnormality, ketogenesis and ketolysis, cyclin-dependent kinase C signaling abnormality, and amyloid processing that were significantly associated with invasive characteristics of invasive NFPA. Those data demonstrate that proteomic variations exist between invasive and non-invasive NFPAs. 2DE-based comparative proteomics is an effective approach to identify proteomic variations and pathway network variations. Those findings will serve as a basis to understand the molecular mechanisms of invasive NFPAs and to discover protein markers to effectively manage patients with invasive NFPAs.

Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism.

Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca(2+)-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.

Synaptotagmin I delays the fast inactivation of Kv1.4 channel through interaction with its N-terminus.

BACKGROUND: The voltage-gated potassium channel Kv1.4 is an important A-type potassium channel and modulates the excitability of neurons in central nervous system. Analysis of the interaction between Kv1.4 and its interacting proteins is helpful to elucidate the function and mechanism of the channel. RESULTS: In the present research, synaptotagmin I was for the first time demonstrated to be an interacting protein of Kv1.4 and its interaction with Kv1.4 channel did not require the mediation of other synaptic proteins. Using patch-clamp technique, synaptotagmin I was found to delay the inactivation of Kv1.4 in HEK293T cells in a Ca2+-dependent manner, and this interaction was proven to have specificity. Mutagenesis experiments indicated that synaptotagmin I interacted with the N-terminus of Kv1.4 and thus delayed its N-type fast inactivation. CONCLUSION: These data suggest that synaptotagmin I is an interacting protein of Kv1.4 channel and, as a negative modulator, may play an important role in regulating neuronal excitability and synaptic efficacy.

Physiological and biochemical characterization of egg extract of black widow spiders to uncover molecular basis of egg toxicity

BACKGROUND: Black widow spider (L. tredecimguttatus) has toxic components not only in the venomous glands, but also in other parts of the body and its eggs. It is biologically important to investigate the molecular basis of the egg toxicity. RESULTS: In the present work, an aqueous extract was prepared from the eggs of the spider and characterized using multiple physiological and biochemical strategies. Gel electrophoresis and mass spectrometry demonstrated that the eggs are rich in high-molecular-mass proteins and the peptides below 5 kDa. The lyophilized extract of the eggs had a protein content of 34.22% and was shown to have a strong toxicity towards mammals and insects. When applied at a concentration of 0.25 mg/mL, the extract could completely block the neuromuscular transmission in mouse isolated phrenic nerve-hemidiaphragm preparations within 12.0 ± 1.5 min. Using whole-cell patch-clamp technique, the egg extract was demonstrated to be able to inhibit the voltage-activated Na+, K+and Ca2+ currents in rat DRG neurons. In addition, the extract displayed activities of multiple hydrolases. Finally, the molecular basis of the egg toxicity was discussed. CONCLUSIONS: The eggs of black widow spiders are rich in proteinous compounds particularly the high-molecular-mass proteins with different types of biological activity The neurotoxic and other active compounds in the eggs are believed to play important roles in the eggs' toxic actions.

Purification and partial characterization of a novel neurotoxic protein from eggs of black widow spiders (Latrodectus tredecimguttatus).

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse-isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin-sensitive sodium current in rat dorsal root ganglion cells. The N-terminal sequence of the protein was identified by the Edman degradation to be N-S-I-A-D-D-R-Y-R-W-P-G-Y-P-G-A-G-L-I-P-Y-I-I-D-S-. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin-I) is a novel neurotoxic protein purified from the eggs of black widow spiders.